Increased Distraction Rates Influence Precursor TissueComposition Without Affecting Bone Regeneration*

MARK RICHARDS, KENNETH M. KOZLOFF, JAMES A.GOULET, and STEVEN A. GOLDSTEIN

ABSTRACT

The effect of increased distraction rate on bony tissue differentiation was studied using a paired bilateralmodel of rat femur lengthening. After a 6-day latency period, one randomly selected femur for each rat wasdistracted at 0.5 mm/day (normal rate) for 12 days, and the contralateral femur was distracted at 1.5 mm/day(increased rate) for 4 days. Femoral lengthening for each side was 6.0 mm, leaving the increased rate leg withan extra 8 days of consolidation compared with the normal rate limb. Group I rats (n 5 9) were killed at day18 postsurgery and analyzed for cartilage tissue composition and distribution. Group II rats (n 5 7) werekilled on day 36 postsurgery and analyzed by three-dimensional microcomputed tomography (MCT) forchanges in new bone volume. Digital color analysis of slides stained with type II collagen antibody showedincreases in cartilaginous tissue formation on the increased rate side (1.51 mm2 vs. 0.83 mm2; p 5 0.10). Nodifferences in new bone volume were detected between increased rate limbs and their contralateral controls(46.13 mm3 vs. 42.69 mm3; p 5 0.63). These findings suggest that intermediate distraction rates may influenceprecursor tissue composition without affecting the final amount of new bone formed. Because damage to thetissue was not detected at either time point, these changes in chondrogenesis may reflect sensitivity of thepluripotential gap tissue to tension accumulation during lengthening. Future work with this in vivo model isfocused on improving our understanding of the mechanisms behind this strain sensitivity. (J Bone Miner Res2000;15:982–989)

Key words: distraction osteogenesis, chondrogenesis, bone regeneration, strain, damage

INTRODUCTION

DISTRACTION OSTEOGENESIS(DO) is an increasingly pop-ular technique used to stimulate new bone formation to

treat orthopedic disorders resulting from bony defects anddeficits. Distraction can be performed on a variety of bones,from the femur to the mandible, and lengthenings of greaterthan 10 cm have been reported (J.A. Goulet, personal com-munication, 1996).(1–3) One principle generally accepted ascritical to the technique’s success is the separation of thetwo bony ends at a slow, controlled rate of about 1 mm/day.

Ilizarov, who pioneered most clinical applications of DO,repeatedly stressed the importance of “slow, steady trac-tion” for successful bone regeneration.(1,4,5)However, fasterdistraction rates might reduce the amount of time requiredfor lengthening and subsequent consolidation. Shorter treat-ment times also might decrease the incidence of pin siteinfections and other complications.(2,3) This has promptedorthopedic surgeons to ask whether distraction rates mightbe increased to shorten treatment time.

Most of the evidence supporting a rate of 1 mm/day hascome from clinical outcome studies. Often, these results aresimply observations that this rate was successful, and pos-sible confounding factors are not accounted for in thesereports.(1,6–8)The failure of the Wagner technique to bridge

*Presented in part at the ASME Summer Biomedical Engineer-ing Meeting, Sun River, Oregon, U.S.A., 1997.

Orthopaedic Research Laboratories, University of Michigan, Ann Arbor, Michigan, U.S.A.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 15, Number 5, 2000© 2000 American Society for Bone and Mineral Research

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spontaneously the gap with bone after distracting the twobony ends as quickly as the soft tissues will allow, has beenwell documented.(9) Ilizarov, in his classic canine experi-ments, showed that increasing the distraction rate from 1.0to 2.0 mm/day was detrimental to gap tissue ossification.(5)

However, these results were largely qualitative, and samplesizes within the many different groups were small.

Several investigators have studied how tension accumu-lates within the distraction gap during lengthening.(7,8,10)

Rapid tension accumulation has been postulated to contrib-ute to decreased healing at higher distraction rates. This issupported by the clinical observation that joint contractures,a common complication of limb lengthening, are amelio-rated by stopping distractions for a few days.(3) Recent workby our group has quantified the viscoelastic nature of gaptissue to predict tension occurring during the distractionprocess.(11) However, cases also have been reported inwhich distraction rates had to be increased in order to avoidpremature consolidation. This is especially true for youngerpatients and for lengthenings over intramedullary nails (J.A.Goulet, personal communication, 1996).(12)

This body of evidence raises several important questions.Do modest increases in distraction rate prove detrimental tobone regeneration? Do they affect precursor tissue compo-sition within the gap? If so, what are the mechanismsresponsible for these changes? Answers to these issueswould likely improve our understanding of how physicalforces and strains influence bone repair and regeneration atboth the tissue and the organ levels. Answers to thesequestions also might allow surgeons to tailor distractionprotocols to optimize patient care. The objectives of thisstudy were to determine whether increases in distractionrate (1) affect new bone volume and (2) alter the amount ordistribution of cartilaginous tissue within the distractiongap. This article presents paired data from a bilateral ratfemoral distraction model. Rats were killed at both an earlyand a late time point to study the effects of increaseddistraction rate on both the soft and the mineralized tissuesthat contribute to bony healing during limb lengthening.

MATERIALS AND METHODS

Surgery, distraction protocol, and euthanasia

Bilateral lengthening of femora in male Sprague-Dawleyrats, 1 year in age and 500 g in weight, was performed. Allprocedures were conducted under general anesthesia andsterile conditions. Using a custom drill guide, four threadedSteinmann pins, 0.067 in. in diameter, were placed in theanterolateral aspect of both femora after percutaneouspredrilling. A custom, monolateral external distraction de-vice was then placed over the pins and locked into place(Fig. 1). The diaphysis of the femur was then exposed via asmall anterolateral incision and blunt dissection through theoverlying tissues. After elevation and protection of theperiosteum, the femur was transected using an oscillatingsaw with saline irrigation. The wound site was cleaned ofbone debris, and the distraction gap was closed, bringing thetwo cortical bony ends into contact. Subcutaneous tissueand skin were closed with 3.0 vicryl suture and staples,respectively. All animals were fully weight bearing less than1 h after surgery and were given chow and water ad libitumuntil the time of death. Pin sites were cleaned daily withantibiotic solution. Weekly radiographs were taken to mon-itor animals for the presence of fractures, pin site infections,or malreduction of the bony ends (Fig. 2).

All rats in this study followed the same distraction pro-tocol. After a 6-day latency period, one randomly chosenfemur was distracted 0.50 mm/day (0.25 mm, two timesdaily) for 12 days. The contralateral femur was distracted atthree times this rate (0.75 mm, two times daily) for 4 days.Distraction for both legs began on the same day after the6-day latency period. The total femoral lengthening was 6.0mm on both sides and was completed on day 10 postsurgery

FIG. 1. Custom external monolateral distraction devicefor lengthening of rat femora.

FIG. 2. Series of radiographs at (A) 14 days postsurgery,(B) 21 days postsurgery, and (C) 28 days postsurgery,illustrating the distraction of the rat femora. The fixatordevice was very stable, as evidenced by excellent alignmentbetween the two bony ends throughout the lengtheningprocedure.

983INCREASED DISTRACTION RATES INFLUENCE PRECURSOR TISSUES

for the increased rate leg and day 18 postsurgery on thecontralateral limb. Rats were divided into two groups.Group I rats (n 5 9) were killed on day 18, immediatelyafter lengthening was finished, to study soft tissue develop-ment within the distraction callus. Group II rats (n 5 7)were killed on day 36 to determine mineralized bone vol-umes within the gap. Rats were killed by carbon monoxideasphyxiation. Limbs were removed by disarticulation andstripped of muscles and other superficial tissues. Distractionzone tissue was harvested with the fixators and neighboringcortical bone intact to ensure it was not damaged duringprocessing. Gap tissue was immediately fixed in cold 10%neutral buffered formalin (NBF) for 48 h. Afterward, sam-ples were placed in cold 70% ethanol while awaiting furtheranalysis. The University Committee on Use and Care ofAnimals approved all procedures at The University ofMichigan, in accordance with National Institutes of Health(NIH) guidelines.

Microcomputed tomography

While in ethanol, distraction zones from Group II rats(day 36) were scanned by three-dimensional microcom-puted tomography (MCT) at a resolution of 50mm/voxel(Fig. 3).(13) MCT images were reconstructed at a byte rangesuch that a voxel intensity of 255 corresponded to fullymineralized cortical bone. From these images, histograms ofvoxel count versus voxel intensity within the distraction gapwere constructed. Voxel counts were obtained in two re-gions of interest: (1) gap only tissue (BVGap), taken astissue within the gap bounded by the radial dimensions ofthe original cortex; (2) BVFull, an extended count, whichincluded the entire distraction callus. This technique haspreviously been used to determine new bone volumes in arabbit model of DO.(12,14)Information regarding soft tissueswithin the gap cannot be obtained from MCT images. Dis-traction zones from group I rats (day 18) possessed little orno mineralized bone within the gap and, therefore, were notscanned.

Histology

Group I rats were decalcified and embedded in paraffinusing standard histological techniques. Seven-micrometer-thin longitudinal sections were taken through the distractiongap and stained with toluidine blue (1%) and safranin-O(1.0%)/fast green (0.02%) for qualitative analysis underlight microscopy. A second set of slides were stained withtype II antibody (II-II6B3)(15) for detection of cartilagewithin the distraction gap. Briefly, slides were hydrated andincubated with hyaluronidase (5 mg/ml) for 1 h at 37°C.Methanol/peroxidase quenching was performed to diminishendogenous peroxidase activity. After blocking with 10%bovine serum albumin (BSA) solution, slides were incu-bated with a 1:300 dilution of the antibody for 2 h at37°C.Primary antibody was detected using a commercial biotin-ylated secondary antibody and chromagen kit (Zymed,South San Francisco, CA, U.S.A.). No counterstaining wasperformed to enhance contrast between regions of positiveand negative staining. Rabbit and rat growth plates wereused as positive controls; slides stained with no primaryantibody served as negative controls.

Group II rats were dehydrated and embedded undecalci-fied in polymethyl methacrylate (PMMA) (after MCT scan-ning) in a specific known orientation. Following a previ-ously described protocol,(16) embedded blocks were thick-sectioned at 1000mm using a diamond-blade saw (EXAKT,Oklahoma City, OK, U.S.A.). Thick sections were mountedon radiolucent plastic, polished, and surface-stained withtoluidine blue for qualitative histological analyses (Fig. 4),using an established histological protocol.(17)

Digital color analysis

Digital color analysis was performed to quantify areas ofcartilaginous tissue on antibody-stained slides from group Irats. Color images of two midsagittal slides from eachdistraction zone in each rat were captured (Javelin, Tor-rance, CA, U.S.A.; NIH Image, Bethesda, MD, U.S.A.).

FIG. 3. Three-dimensional MCT image of rat distractionzone (resolution, 50mm/voxel). Consolidation was notachieved at 36 days postsurgery in either increased rate(left) or normal rate (right) femora. FIG. 4. Micrograph showing new bone formation within

the distraction gap. New bone in periosteal locations oftenlacked trabecular features (top). Cortical bone surface canbe seen at left. Toluidine blue, magnification,34.

984 RICHARDS ET AL.

Images were thresholded to separate positive-staining car-tilaginous pixels from other pixels and counted (MATLAB,Mountain View, CA, U.S.A.) to determine the area ofcartilaginous tissue within the gap. Image resolutionsranged from 20 to 30mm/pixel (Fig. 5). Paired distractionzones (and their respective slides) were obtained, processed,stained, and imaged simultaneously to increase the consis-tency of paired analyses. All slides were examined carefullyto ensure that only tissue that possessed the morphologicalfeatures of cartilage was counted in the color analysis.

Statistical analysis

Paired t-tests were performed to detect differences inoutcome variables between paired rats in groups I and II(SYSTAT, Evanston, IL, U.S.A.). Results were consideredsignificant at a level ofp # 0.05.

RESULTS

Surgical results

Two rats were lost during surgery to intraoperative frac-tures of the femur. Otherwise, rats tolerated surgery and theincreased rate of distraction well. A predisposition to pinsite infections was observed for some of the group II ratsbecause of their longer time of study. However, none ofthese infections caused pin loosening or compromised thetissue within the distraction gap. Rats resumed normal cageactivity less than 1 h after surgery; it should be stressed thatat no time did animals appear to favor one limb over anotherin this bilateral model.

MCT and new bone volume

Three-dimensional MCT images indicated that bone re-generation was most prevalent in periosteal locations (Fig.3). Often, this new periosteal bone appeared compact and

lacked trabecular features. Histograms obtained from MCTimages exhibited a bimodal distribution that separated min-eralized and nonmineralized voxels within the distractiongap tissue. This separation between the two voxel popula-tions was present in histograms constructed for all animalskilled at 36 days. Both the BVGap and the BVFull measuresof new bone volume were increased slightly in rat femoradistracted at the higher rate (Fig. 6). However, these differ-ences were small and not statistically significant (BVGap,46.13 mm3 [1.5 mm/day] vs. 42.69 mm3 [0.5 mm/day],p 50.63; BVFull, 82.21 mm3 [1.5 mm/day] vs. 75.05 mm3 [0.5mm/day],p 5 0.53).

Histology

Light microscopy analysis of sections from groups I andII stained with toluidine blue confirmed that alignment ofthe two bony ends was maintained throughout distraction(Fig. 2). Only small amounts of differentiated tissue werepresent in the gap center at 18 days, particularly near themarrow at proximal and distal ends. Abundant fibrous tissuewas seen outside the gap in periosteal regions, giving theappearance of a large fracture callus. More tissue prolifer-ation generally was seen on the side opposite the distractor.Cartilage was seen in all samples in varying amounts, indi-cating that bone was likely forming by both endochondraland intramembranous mechanisms. Tissue orientation alongthe applied tension vector of the bone was evident, espe-cially in samples distracted at the increased rate. In agree-ment with the MCT data from group II rats, new boneformation activity at 36 days was focused in periostealregions of the distraction gap (Fig. 4). Some of this boneappeared lamellar, mature, and lacked any trabecular fea-tures. In contrast, regions in the center of the distraction gapoften showed little evidence of mineralized bone formation.Rigorous morphological analyses of trabecular bone archi-

FIG. 5. Areas of tissue staining positive for type II col-lagen in both the entire region (Full) and the strict distrac-tion gap (Gap) are shown. Increased distraction rates in-duced increases in staining when periosteal regions wereincluded in image analysis (Full; 1.51 mm2 vs. 0.83 mm2;p 5 0.10).

FIG. 6. New bone volume increased slightly when dis-traction rate was raised from 0.5 to 1.5 mm/day in both theentire region (BVFull) and the strict distraction gap (BV-Gap). These differences were small and not statisticallysignificant.

985INCREASED DISTRACTION RATES INFLUENCE PRECURSOR TISSUES

tecture could not be performed because an inadequate num-ber of fields containing trabecular bone were present.

Digital color analysis

Digital color analysis of group I rats showed differencesin the amount of cartilage between femora distracted at thetwo different rates. Analyses of slides stained with type IIantibody indicated a nearly 2-fold increase in cartilaginoustissue area (1.51 mm2 vs. 0.83 mm2, p 5 0.10; Fig. 5). Thistrend did not achieve statistical significance.

DISCUSSION

Optimal distraction rates for leg lengthening have beendefined largely in the clinical arena to lie somewhere be-tween two undesirable extremes. At low distraction rates,premature consolidation occurs, and the lengthening goal isnot achieved. On the other hand, at faster rates, the distrac-tion gap and surrounding tissues might be damaged, hin-dering new bone formation. However, the true effect ofdistraction rate on bone regeneration is not well understood.In this study, increased rates of lengthening were imple-mented in a bilateral rat model of distraction to investigatetheir effect on bone regeneration. In addition, gap tissue inthe rat model before mineralization was studied to detectchanges in cartilaginous tissue differentiation. In this fash-ion, the effects of increased stretch (and associated increasesin tension levels) on bone regeneration at two different timepoints could be studied and correlated with one another.

Higher distraction rates were associated with increases incartilage formation at 18 days postsurgery. Staining for typeII collagen was more reliable than the safranin-O technique;however, both methods showed the observed increase inchondrogenesis. The presence of cartilage was always as-sociated with bone formation activity and usually was in aperiosteal location. Thus, the majority of tissue differentia-tion at this early stage of bone repair appeared to be occur-ring in periosteal regions of the gap, in agreement withobservations in other fracture and distraction models.(4,18–22)

The 3-fold increase in distraction rate had no effect on newbone volumes at 36 days postsurgery; thus femora distractedat both rates appeared to make bone at similar rates.

It should be stressed that these experiments were notcarried out to the time point at which bony bridging of thegap occurred. Two rats killed at 64 days postoperativelydisplayed nearly complete bridging of the gap (unpublisheddata, Richards and Goldstein, 1997), indicating that themodel is successful and does not induce pseudarthrosisformation. It is not likely that differences in new bonevolume between the normal and increased rate legs wouldappear between the 36-day and 64-day time points, but thiscannot be ruled out based on the data presented here.

In this study, the use of an oscillating saw to produce theosteotomy, rather than a more clinically recognized corti-cotomy, was chosen to produce a uniform distraction gapgeometry and to avoid the oblique fractures often seen incorticotomies.(20) Some authors claim that corticotomiesprovide a faster healing response(18) by maintaining the

medullary blood vessels intact, in comparison with an os-teotomy made all the way through the marrow cavity. Oth-ers see no evident difference in healing patterns between thetwo.(19) Frierson advises against the use of an oscillatingsaw for osteotomies, because of an observed delayed con-solidation in dogs.(20) Despite the use of copious salineirrigation throughout the osteotomy in the present study, itis possible that the temperature increased at the osteotomysite during the procedure. This could, in theory, causethermal damage, which could, in turn, lead to cartilageformation within the distraction gap. However, it is unlikelythat this mechanism is responsible for the observed increasein cartilage seen in the increased rate leg of the animalcompared with the normal rate leg, because the osteotomyprotocol was the same regardless of distraction rate.

Various mechanisms might be advanced to account forthe increases in cartilage formation, many of which centeraround damage. Increased distraction rates might lead tohigher peak stresses and strains immediately after distrac-tion; likewise, more rapid tension accumulation during thefull course of lengthening may occur at higher distractionrates. Such patterns of tension accumulation have beendocumented in both clinical studies and work describing theviscoelastic properties of gap tissue.(8,11) These alteredphysical forces and strains then likely exert their influenceon important biological processes. Damage to newly form-ing blood vessels, the periosteum, and/or scaffolding ele-ments within the distraction gap might decrease vascularityor oxygen tension to the tissues, rendering them more ame-nable to cartilage differentiation than other tissue types.(23)

Existing blood vessels may become damaged at high dis-traction rates, further reducing the overall blood supply tothe wound.(23) However, it cannot be ruled out that thetissue may be stretched at a rate that simply exceeds thecapacity to generate blood vessels, causing a deficit of bloodsupply within the gap. Li and coworkers have found thathigh rates of distraction inhibit or reduce angiogenesiswithin a rabbit model of DO.(24) At rates of 0.7 mm/day and1.3 mm/day, newly formed bone was always associatedwith blood vessels. Additionally, at rates above 0.7 mm/day,cartilage islands were found within the tissue and were notassociated with angiogenic markers. A similar phenomenoncould account for the higher cartilage formation seen in thepresent experiment on the legs distracted at the increasedrate of 1.5 mm/day.

Although damage to the tissue within the distraction gapmay have influenced the resulting tissue type, it also ispossible that the observed increase in chondrogenesis wasthe result of local strain influences on fundamental pro-cesses of differentiation from pluripotential mesenchymaltissue. Pauwels, Carter, and others have hypothesized thatlocal strain environment influences cellular events, includ-ing differentiation and gene expression.(25–28) Specifically,they have argued that high distortional strains favor fibroustissue formation, whereas more “quiescent” strain regionsfavor the formation of cartilage. Evidence to support theexistence of such a mechanism comes from the cellularbiology literature.(29–31)Experiments have shown that chon-drocytes grown on adherent media “dedifferentiate” andbegin to express type I collagen as they assume a more

986 RICHARDS ET AL.

elliptical shape. Resuspension of these cells in media al-lowed the chondrocytes to reassume their spherical shape, atwhich time they again expressed type II collagen.

Thus, it has been argued that strains with a high distor-tional character might discourage chondrogenesis. On asuperficial level, these views are not consistent with thefindings in this study. As stretches are imposed along asingle axis, it might be expected that increased distractionrates would lead to increased distortion of the tissue, espe-cially in the direction of applied step elongations. Such atheory would argue for increased fibrous tissue formation insuch a mechanical environment. However, in this study,increased distraction rates appeared to favor chondrogenesisand not fibrous tissue formation. It should be emphasized,though, that information regarding a local strain environ-ment was not rigorously established for changes in distrac-tion rate in this animal model. This would require nonlinear,three-dimensional, viscoelastic finite element analyses, be-yond the scope of the present study.

Strain-related inhibition of chondrogenesis has been pos-tulated to occur in another rat distraction model, in whichthe formation of cartilage within a distraction gap was onlyobserved after the distraction protocol was halted.(32) In thisstudy, rat femora were distracted 0.165 mm every 12 h. Atthis slower distraction rate, the gap was bridged completelyby bone when the final length of 7 mm was obtained, and atthis point, no cartilage was observed. After 21 days ofconsolidation, some parts of the distraction gap showedcolumns of chondrocytes and cartilage, suggesting endo-chondral bone formation. The lack of cartilage formationduring distraction was attributed to the generation of tensiledistraction forces. However, it is also possible that this lowdistraction rate (0.33 mm/day) reduced tension accumula-tion and potential damage within the gap. This mechanicalenvironment may then have been more permissive of fi-brous tissue and new bone formation.

Although increased distraction rates did influence carti-laginous tissue production, they did not have any effect onnew bone volume at 36 days postsurgery in this animalmodel. No differences in bone volume were detected be-tween the normal and increased distraction rate femora atthis time point. Thus, new bone volume was unaffected byany potential prior differences in cartilaginous tissue forma-tion. This finding is different from those of Aronson et al.,who qualitatively described improved mineralized bone for-mation with tibial lengthenings of 0.5 mm/day, comparedwith 2.0 mm/day.(33) There are several important differ-ences between the current study and that of Aronson et al.that might explain this discrepancy. Their model was of rattibial lengthening; because the femur has a better bloodsupply than the tibia, this could impact the results here.Studies by Aronson lengthened only one limb in each ani-mal and used a ring fixator; clearly, this could create verydifferent strain environments within the developing gaptissue. Furthermore, the described changes in new boneformation were not quantified, and their increased rate wasone-third faster than that used in our protocol.(33)

Nonrecoverable tissue damage has been shown to exist inhigh distraction rate models, in which necrotic tissue andcysts remained within the distracted tissue after 4 weeks of

consolidation.(23) This could suggest that increases above acertain distraction rate result in excessive and irreparabledamage to the tissue. In the current study, increased distrac-tion rates caused greater cartilage formation at an early timepoint but no change in total new bone volume 18 days later.Furthermore, no evidence of necrosis or cysts was seen inany of the increased rate distraction gap tissue samples at 18days or 36 days. Because of the bilateral nature of ouranimal model, the increased rate limb had an extra 8 days ofconsolidation compared with the normal rate limb. Al-though the higher distraction rate initially may have causeddamage reflected by the increased cartilage levels, it ispossible that the increased time of consolidation allowedmore healing to occur in the high rate limb. Increasedendochondral bone formation may have contributed to thelevels of bone found at the final time point. In addition, ourdistraction rates were higher than the rates of Jazrawi et al.(1.3 mm/day), who saw no cartilage formation during thedistraction, and lower than the rates of Aronson (2.0 mm/day) and Li (2.7 mm/day).(23,32,33)Our observations supportthe currently held view that an intermediate distraction rateis optimal. By increasing distraction rate to a value that mayincrease cartilage formation but not cause permanent tissuedamage nor hinder new bone formation, the final desiredlength might be reached at an earlier time point. However,additional consolidation time may then be required in orderto achieve required bony filling of the gap.

This raises an important question in its own right. Formore than a decade, scientists and clinicians have arguedover the role cartilage plays during bone regeneration dur-ing distraction. Some practitioners have claimed that boneformation during distraction should be entirely intramem-branous. Any cartilage within the distraction gap was as-sumed to result from either poor fixator stability (and ex-cessive micromotion) or failure to preserve the blood supplyduring surgery.(1,4,5,34) Molecular analyses of distractiongap tissue have supported this viewpoint.(35,36) However,reports of cartilaginous tissue within the distraction gaphave become increasingly common in both ring and mono-lateral fixator systems.(18–22,37)Thus, although these animalmodels of distraction provide an excellent means for thestudy of tissue differentiation in response to a wide varietyof stimuli, it is possible that during distraction, the degree ofbony healing does not depend on the mechanism of itsformation.

From a strictly clinical standpoint, it is important torealize that the highest achievable distraction rate dependsnot only on the response of the newly forming bony tissuebut also on constraints placed on it by surrounding tissues.Numerous reports have documented an increased incidenceof joint contractures and angular deformities at higher dis-traction rates,(7,10,38) likely because of an inability of sur-rounding muscles to stretch adequately. Concerns regardingthe ability of nerves to adapt to high distraction rates alsohave been raised.(39) Thus, the effects of increased distrac-tion rate on differentiation of bony precursor tissue and newbone volumes presented here should be appreciated in thislarger clinical context. The results of this study, whichsupport the use of an intermediate distraction rate, are inagreement with current clinical views and practice.

987INCREASED DISTRACTION RATES INFLUENCE PRECURSOR TISSUES

Finally, certain specifics of the animal model merit com-ment. First, the bone lengthened in this study was the femur.Larger animal models typically have lengthened the tibia,because fixators are more easily placed on the distalleg.(1,4,5,18–21,34)The femur has a much better blood supplythan the tibia, and clinical results support the contention thathealing occurs more rapidly and reliably in the femur thanin the tibia.(40–42) As mentioned before, this may accountfor discrepancies between the results presented here andthose of Aronson.(33) Second, species specificity could con-tribute to the observed insensitivity of bone formation toincreased distraction rate in this model. Rats are very robustanimals and their growth plates do not close; therefore, theymay respond to the challenge of bone regeneration morevigorously than other animals. Current work with this ratmodel of DO is underway to address these issues and furtherexplore the mechanisms by which increased distractionrates influence chondrogenesis and bone regeneration.

ACKNOWLEDGMENTS

The authors thank A.R. McDonald, E. Alsberg, T. Henne,M.J. Hernandez, V. Bhatia, C. DeBano, J.P. Rouleau, M.Stock, D.C. Kayner, K.A. Sweet, and B. Nolan for helpingto make this work possible. Statistical consultations wereprovided by Prof. M.B. Brown, Department of Biostatistics.Support of this work by The Orthopedic Trauma Associa-tion, NIH (AR-20557), The Orthopedic Research and Edu-cation Foundation, The Whitaker Foundation, and The Or-thopaedic Research Labs, University of Michigan isgratefully acknowledged.

REFERENCES

1. Ilizarov GA 1990 Clinical application of the tension-stresseffect for limb lengthening. Clin Orthop Rel Res250:8–26.

2. Paley D 1988 Current techniques of limb lengthening. J PedOrthop8:73–92.

3. Paley D 1990 Problems, obstacles, and complications of limblengthening by the Ilizarov technique. Clin Orthop Rel Res250:81–104.

4. Ilizarov GA 1989 The tension-stress effect on the genesis andgrowth of tissues. Part I.The influence of stability of fixationand soft-tissue preservation. Clin Orthop Rel Res238:249–281.

5. Ilizarov GA 1989 The tension-stress effect on the genesis andgrowth of tissues. Part II. The influence of the rate and fre-quency of distraction. Clin Orthop Rel Res239:263–285.

6. Cattaneo R, Villa A, Catagni MA, Bell DF 1990 Lengtheningof the humerus using the Ilizarov technique: Description of themethod and report of 43 cases. Clin Orthop Rel Res250:117–124.

7. Dwyer JSM, Owen PJ, Evans GA, Kuiper JH, Richardson JB1996 Stiffness measurements to assess healing during leglengthening. J Bone Joint Surg78B:286–289.

8. Younger ASE, Mackenzie WG, Morrison JB 1994 Femoralforces during limb lengthening in children. Clin Orthop RelRes301:55–63.

9. Wiedemann M 1996 Callus distraction: A new method? ClinOrthop Rel Res327:291–304.

10. Simpson AHRW, Cunningham JL, Kenwright J 1996 Theforces which develop in the tissues during leg lengthening.J Bone Joint Surg78B:979–983.

11. Richards M, Wineman AS, Alsberg E, Goulet JA, GoldsteinSA 1999 Viscoelastic characterization of mesenchymal gaptissue and consequences for tension accumulation during dis-traction. J Biomech Eng121:116–123.

12. Richards M, Goulet JA, Weiss JA, Waanders NA, SchafflerMB, Goldstein SA 1998 Bone regeneration and fracture heal-ing: Experience with the distraction model. Clin Orthop RelRes355S:191–204.

13. Goulet RW, Goldstein SA, Ciarelli MJ, Kuhn JL, Brown MB,Feldkamp LA 1994 The relationship between the structuraland orthogonal compressive properties of trabecular bone.J Biomech27:375–389.

14. Richards M, Waanders NA, Weiss JA, Bhatia V, Senunas LE,Schaffler MB, Goldstein SA, Goulet JA 1999 Reduced gapstrains induce changes in bone regeneration during distraction.J Biomech Eng121:348–355.

15. Chen Q, Fitch JM, Gibney E, Linsenmayer TF 1993 Type IIcollagen during cartilage and corneal development. Immuno-histochemical analysis with an anti-telopeptide antibody. DevDyn 196:47–53.

16. Richards M, Goulet JA, Schaffler MB, Goldstein SA 1999Temporal and spatial characterization of regenerate bone in thelengthened rabbit tibia. J Bone Miner Res 14 :1978–86.

17. Sterchi D, Eurell J 1989 A new method for preparation ofundecalcified bone sections. Stain Tech64:201–205.

18. Brutscher R, Rahn BA, Ruter A, Perren SM 1993 The role ofcorticotomy and osteotomy in the treatment of bone defectsusing the Ilizarov technique. J Orthop Trauma7:261–269.

19. Delloye C, Delefortrie G, Coutelier L, Vincent A 1990 Boneregenerate formation in cortical bone during distraction length-ening. Clin Orthop250:34–42.

20. Frierson M, Ibrahim K, Boles M, Bote H, Ganey T 1994Distraction osteogenesis: A comparison of osteotomy tech-niques. Clin Orthop Rel Res301:19–24.

21. Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y 1988Bone lengthening in rabbits by callus distraction: The role ofendosteum and periosteum. J Bone Joint Surg70B:543–549.

22. Shapiro F, Rand F, Upton J, Barone L 1992 Histologic patternsof bone formation in rabbit distraction osteogenesis. TransOrtho Res Soc38:585.

23. Li G, Simpson A, Kenwright J, Triffit J 1997 Assessment ofcell proliferation in regenerating bone during distraction os-teogenesis at different distraction rates. J Orthop Res15:765–772.

24. Li G, Simpson A, Kenwright J, Triffit J 1999 Effect of length-ening rate on angiogenesis during distraction osteogenesis.J Orthop Res17:362–367.

25. Carter DR, Giori NJ 1989 Effect of mechanical stress on tissuedifferentiation in the bony implant bed. In: Davies JE (ed.) TheBone-Biomaterial Interface. University of Toronto Press, To-ronto, Canada, pp. 367–379.

26. Carter DR 1987 Mechanical loading history and skeletal biol-ogy. J Biomech20:1095–1109.

27. Giori NJ, Beaupre GS, Carter DR 1993 Cellular shape andpressure may mediate mechanical control of tissue composi-tion in tendons. J Orthop Res11:581–591.

28. Pauwels F 1980 Biomechanics of the Locomotor Apparatus.Springer-Verlag, Berlin.

29. Benya PD, Brown PD, Padilla SR 1988 Microfilament mod-ulation by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differentiated colla-gen phenotype without a change in shape. J Cell Biol106:161–170.

30. Benya PD, Shaffer JD 1982 Dedifferentiated chondrocytesreexpress the differentiated collagen phenotype when culturedin agarose gels. Cell30:215–224.

31. Brown PD, Benya PD 1988 Alterations in chondrocyte cy-toskeletal architecture during phenotypic modulation by reti-

988 RICHARDS ET AL.

noic acid and dihydrocytochalasin B-induced reexpression.J Cell Biol 106:171–179.

32. Jazrawi L, Majeska R, Klein M, Kagel E, Stromberg L, Ein-horn T 1998 Bone and cartilage formation in an experimentalmodel of distraction osteogenesis. J Orthop Trauma12:111–116.

33. Aronson J, Shen X, Skinner R, Hogue W, Badger T, LumpkinC 1997 Rat model of distraction osteogenesis. J Orthop Res15:221–226.

34. Aronson J, Good B, Stewart C, Harrison B, Harp JH 1990Preliminary studies of mineralization during distraction osteo-genesis. Clin Orthop Rel Res250:43–49.

35. Kallio TJ, Vauhkonen MV, Peltonen JI, Karaharju EO 1994Early bone matrix formation during distraction. Acta OrthopScand65:467–471.

36. Karaharju-Suvanto T, Vauhkonen M 1994 Synthesis of fibril-forming collagen during distraction bone healing in the man-dible. Eur J Exp Musculoskel Res3:145.

37. Windhager R, Tsuboyama T, Siegl H, Groszschmidt K, SeidlG, Schneider B, Plenk H 1995 Effect of bone cylinder lengthon distraction osteogenesis in the rabbit tibia. J Orthop Res13:620–628.

38. Hyodo A, Kotschi H, Kambic H, Muschler G 1996 Bonetransport using intramedullary fixation and a single flexibletraction cable. Clin Orthop Rel Res325:256–268.

39. Kwan MK, Wall EJ, Massie J, Garfin SR 1992 Strain, stressand strech of periperhal nerve: Rabbit experiment in vitro andin vivo. Acta Orthop Scand63:267–272.

40. Bone LB, Johnson KD 1986 Treatment of tibial fractures byreaming and intramedullary nailing. J Bone Joint Surg68:877–887.

41. Court-Brown CM, Christie J, McQueen MM 1990 Closedintramedullary tibial nailing: Its use in closed and type I openfractures. J Bone Joint Surg74:605–611.

42. Wiss DA, Stetson WB 1995 Unstable fractures of the tibiatreated with a reamed intramedullary interlocking nail. ClinOrthop Rel Res315:56–63.

Address reprint requests to:Dr. Steven A. Goldstein

Orthopaedic Research LaboratoriesRoom G-161, 400 NIBUniversity of Michigan

Ann Arbor, MI 48109-0486, U.S.A.

Received in original form February 11, 1999; in revised formNovember 29, 1999; accepted January 3, 2000.

989INCREASED DISTRACTION RATES INFLUENCE PRECURSOR TISSUES